Molecular detection of Coxiella spp. in ticks (Ixodidae and Argasidae) infesting domestic and wild animals: with notes on the epidemiology of tick-borne Coxiella burnetii in Asia

Tick-borne Coxiella spp. are emerging in novel regions infecting different hosts, but information regarding their occurrence is limited. The purpose of this study was the molecular screening of Coxiella spp. in various ticks infesting goats, sheep, camels, cattle, wild mice, and domestic fowls (Gallus gallus domesticus) in various districts of Khyber Pakhtunkhwa, Pakistan. Morphologically identified tick species were confirmed by obtaining their cox1 sequences and were molecularly screened for Coxiella spp. by sequencing GroEL fragments. Almost 345 out of 678 (50.9%) hosts were infested by nine tick species. Regarding the age groups, the hosts having an age >3 years were highly infested (192/345, 55.6%), while gender-wise infestation was higher in female hosts (237/345, 68.7%). In collected ticks, the nymphs were outnumbered (613/1,119, 54.8%), followed by adult females (293/1,119, 26.2%) and males (213/1,119, 19.7%). A total of 227 ticks were processed for molecular identification and detection of Coxiella spp. The obtained cox1 sequences of nine tick species such as Hyalomma dromedarii, Hyalomma anatolicum, Haemaphysalis cornupunctata, Haemaphysalis bispinosa, Haemaphysalis danieli, Haemaphysalis montgomeryi, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Argas persicus showed maximum identities between 99.6% and 100% with the same species and in the phylogenetic tree, clustered to the corresponding species. All the tick species except Ha. danieli and R. microplus were found positive for Coxiella spp. (40/227, 17.6%), including Coxiella burnetii (15/40, 6.7%), Coxiella endosymbionts (14/40, 6.3%), and different Coxiella spp. (11/40, 4.9%). By the BLAST results, the GroEL fragments of Coxiella spp. showed maximum identity to C. burnetii, Coxiella endosymbionts, and Coxiella sp., and phylogenetically clustered to the corresponding species. This is the first comprehensive report regarding the genetic characterization of Coxiella spp. in Pakistan's ticks infesting domestic and wild hosts. Proper surveillance and management measures should be undertaken to avoid health risks.

Tick-borne Coxiella spp. are emerging in novel regions infecting di erent hosts, but information regarding their occurrence is limited. The purpose of this study was the molecular screening of Coxiella spp. in various ticks infesting goats, sheep, camels, cattle, wild mice, and domestic fowls (Gallus gallus domesticus) in various districts of Khyber Pakhtunkhwa, Pakistan. Morphologically identified tick species were confirmed by obtaining their cox sequences and were molecularly screened for Coxiella spp. by sequencing GroEL fragments. Almost out of ( . %) hosts were infested by nine tick species. Regarding the age groups, the hosts having an age > years were highly infested ( / , . %), while gender-wise infestation was higher in female hosts ( / , . %). In collected ticks, the nymphs were outnumbered ( / , , . %), followed by adult females ( / , , . %) and males ( / , , . %). A total of ticks were processed for molecular identification and detection of Coxiella spp. The obtained cox sequences of nine tick species such as Introduction Ticks are hematophagous ectoparasites, actively contributing to transmitting infectious agents to wild and domestic animals and humans (De la Fuente et al., 2017). Numerous ticks act as distinguished vectors and reservoirs for various pathogens, including bacteria causing rickettsiosis, anaplasmosis, Lyme disease, viruses such as Powassan, and protozoan agents such as Theileria spp. and Babesia spp. (De la Fuente et al., 2017;Karim et al., 2017;Rochlin and Toledo, 2020;Ali et al., 2021). Aside from transmitting various infectious agents, ticks are hosts to many endosymbionts and a diversified microbiome (Špitalská et al., 2018).
Among the bacterial genus Coxiella having one pathogenic species, Coxiella burnetii is a Gram-negative obligate intracellular bacterium distributed worldwide except in New Zealand and French Polynesia (Musso et al., 2014;Eldin et al., 2017). Common reservoirs of C. burnetii are domestic mammals, including cattle, sheep, goats, and camels as well as reptiles, birds, and ticks (Anderson et al., 2013;Abdel-Moein and Hamza, 2017), and have the potential to cause query (Q) fever (Musso et al., 2014). The Qfever was reported for the first time in 1935 from Australia as an outbreak of febrile illness with flu-like symptoms (Derrick, 1937), and its causative agent was initially named Rickettsia burnetii, but later on renamed as C. burnetii (Philip, 1948). Coxiella spp. have been isolated from almost 40 tick species, and hence considered as its tick-borne transmission to animals and humans (Eklund et al., 1947;Beaman and Hung, 1989;Duron et al., 2014). Tick species, including Hyalomma dromedarii, Hyalomma anatolicum, Hyalomma scupense, Rhipicephalus microplus, and Rhipicephalus annulatus, may serve as vector reservoirs for the transmission of C. burnetii in Pakistan (Karim et al., 2017). Coxiella-like endosymbionts were also found in various tick species, and an obligatory mutualism between this bacteria and host ticks has been proven (Smith et al., 2015).
Ticks can transmit Coxiella spp. both transovarially and transstadially to their offspring. Infected ticks excrete enormous amounts of Coxiella spp. in their feces, contaminating the skin of host animals and playing a significant role in the spread of Coxiella infection (Cong et al., 2015;Seo et al., 2016). Coxiella burnetii may resist harsh environmental factors, for instance, dry and hot weather, desiccation, and other antiseptics. As it may affect the productive and reproductive abilities, humans and animals could face long-term infection risks (Ullah et al., 2019). Various techniques have been effectively followed for the surveillance of C. burnetii infection. Still, ELISA is considered an effective technique for its serological diagnosis. In combination with sequencing, PCR is believed to be the best technique for the molecular identification and genetic characterization of C. burnetii (Niemczuk et al., 2014;Bontje et al., 2016).
Coxiella spp. have been detected in different tick species, animals, humans, and soil samples in Asia that have been reported in various studies. Coxiella burnetii is the causative agent of Q-fever, one of the ignored zoonoses in developing countries, including Pakistan. To the best of our knowledge, approximately 24 studies from 1955 to 2022 regarding this infection have been reported in Pakistan, and Q-fever in humans, goats, sheep, cattle, buffaloes, as well as rodents has been serologically documented (Ahmed, 1987;Ullah et al., 2019;Ali et al., 2022a;Hussain et al., 2022). The C. burnetii is also considered a soil-borne pathogen as its isolation has been confirmed from soil samples (Shabbir et al., 2015). Due to the information dearth regarding numerous tick-borne pathogens (TBPs) that infect ruminants and other animals in Pakistan, substantial research is required to investigate the genetic composition of various TBPs, specifically Coxiella spp. Hence, this study aimed to molecularly characterize different tick species infesting domestic and wild animals and screen out the associated Coxiella spp. in Pakistan and summarize the association of Coxiella spp. with ticks infesting various hosts in Asia.

Ethical considerations
The Advance Studies and Research Board (ASRB: Dir/A&R/AWKUM/2022/9396) of the Department of Zoology, Abdul Wali Khan University Mardan, Pakistan, approved prior consent for this study. Additionally, permission was taken from the owners of the animals to observe hosts and ticks collection. All the rules regarding animal welfare regulations were followed while handling the animals. were selected for the current study. These study locations have desertic plains, arid plains, arid hilly, humid plains, and hilly areas with variations in their climatic conditions, altitude, and seasons (winter, spring, summer, and autumn). The summer season is comparatively hot and longer in district Lakki Marwat than in other districts; however, snowfall occurs in winter in the districts of Chitral, Mansehra, Buner, Bajaur, Dir Upper, Dir Lower, and Abbottabad (climate-data.org; accessed on 20 February 2023). Goats, sheep, camels, and cattle are the livestock of the region that are intended for producing dairy products and transportation. These transhumant animals move from one place to another within the district for food and natural pastures. Their diet and resources depend upon climatic conditions that remarkably vary spatiotemporally. The Global Positioning System was used for the geographical coordinates of the districts as mentioned above and designed the study map through ArcGIS v 10.3.1 (Figure 1).

Description of the study area and sampling sites
. /fmicb. .

FIGURE
Map showing the collection sites where ticks were collected.

Tick collection and their morphological identification
The herds of goats, sheep, camels, and cattle were visited for tick collection from September 2021 to August 2022. Moreover, wild mice captured by local farmers on agricultural land and domestic fowls (Gallus gallus domesticus) were also examined for tick specimens. Tick specimens were collected manually from different hosts in the study districts. The collected ticks were morphologically identified under a stereomicroscope (SZ61, Olympus, Japan) using available standard morphological keys (Hoogstraal and Kaiser, 1959;Hoogstraal and Varma, 1962;Hoogstraal and Trapido, 1966;Dhanda and Kulkarni, 1969;Kohls et al., 1970;Cerný and Hoogstraal, 1977;Apanaskevich, 2003;Apanaskevich et al., 2008;Ahmad et al., 2022;Ali et al., 2022b). The identified tick species were categorized according to species, gender, and nymph stage or adult stage, and then preserved in 100% ethanol at room temperature before further analyses.

DNA extraction and molecular screening
The preserved ticks were washed with 70% ethanol, followed by their immersion in distilled water for 10 min to eliminate the external contamination, and subsequently dried on a sterile filter paper. Ha. danieli (3 N, 2 F, and 1 M) were randomly selected and used individually for DNA extraction. Each stage of the morphologically identified tick species was individually crushed using sterile scissors to extract genomic DNA through the standard protocol of the phenol-chloroform method (Sambrook et al., 1989).
The whole extracted genomic DNA of each morphologically identified tick species (each stage) was individually used to amplify cox1 fragments by utilizing species-specific primers in a .
/fmicb. .  (Table 1). PCR reaction mixtures were performed in 25 µL, comprised of 1 µL of each primer at a concentration of 10 pmol/µL (first pair of primers in case of GroEL), 8.5 µL PCR water, 2 µL (100 ng/µL) genomic DNA, and 12.5 µL DreamTaq MasterMix (2×) (Thermo Fisher Scientific, Inc., Waltham, MA, USA). However, 2 µL of PCR product from the first PCR amplified reaction was used instead of genomic DNA in the second PCR run (in the case of GroEL) along with the second pair of primers at the same concentration. In each PCR reaction, PCR water was taken as a negative control, while Hyalomma scupense and Rickettsia massiliae DNA were taken as a positive control for ticks and Coxiella, respectively. The amplified products were loaded in 2% agarose gel to observe the expected band through the Gel Documentation System (BioDoc-It TM Imaging Systems, UVP, LLC, Upland, CA, USA). PCR amplified products were purified via GeneClean II Kit (Qbiogene, Il-lkirch, France) following the manufacturer's protocol. The amplified amplicons were sequenced bidirectionally through the Sanger-based sequencing method (Macrogen, Inc., Seoul, South Korea).

Sequences and phylogenetic analyses
The chromatograms of all the obtained sequences were manually observed and trimmed for purification purposes to remove the contaminated and poor reading regions through SeqMan V. 5 (DNASTAR, Inc., Madison, WI, USA). Final trimmed sequences were subjected to Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) at National Center for Biotechnology Information to get the high identity sequences in FASTA format. ClustalW multiple alignments (Thompson et al., 1994) were used to align all the downloaded sequences along with the obtained and selected outgroup sequences in BioEdit Sequence Alignment Editor V.7.0.5 (Raleigh, NC, USA) (Hall et al., 2011). The phylogenetic trees based on partial fragments of cox1 and GroEL were constructed in MEGA-X (Molecular Evolutionary Genetics Analysis)  through the neighbor-joining method (Tamura-Nei model) and the Maximum Parsimony method (Tamura-Nei model) (Tamura and Nei, 1993) with support of 1000 bootstrapping replicons, respectively. The coding fragments (cox1 and GroEL) were aligned using MUSCLE (Edgar, 2004).

Literature search
The literature search was conducted using databases such as PubMed, Google Scholar, and Web of Sciences, to overview the published studies regarding the detection of C. burnetii in different ticks, animals, humans, or soil in Asia. The keywords used for the search were as follows: tick(s), small ruminant(s), livestock, C. burnetii, Coxiellosis, and Q-fever. Combinations of the aforementioned various keywords were used to retrieve full-text research articles, review articles, short communications, and conference papers. Reference lists of retrieved articles were screened to identify relevant articles (accessed on 16 April 2023) ( Table 2).

Screening of Coxiella in various ticks
Extracted DNA of 227 (20.3%) was used to amplify the fragments of GroEL of Coxiella spp. A total of 40/227 (17.6%) ticks were found positive for Coxiella spp., including C. burnetii Hy. dromedarii

Phylogenetic analyses of the obtained sequences
All amplified PCR products were separately sequenced. The identical sequences were considered as a single consensus sequence. Trimmed and purified sequences of cox1 fragments were obtained from nine tick species, including Ha. cornupunctata, Ha. bispinosa, Ha. danieli, Ha. montgomeryi, Hy. anatolicum, Hy. dromedarii, R. haemaphysaloides, R. microplus, and A. persicus.
The BLAST results showed that the cox1 fragments of the nine tick species were 99.6-100% identical to the corresponding species. Additionally, these sequences were phylogenetically clustered to the corresponding species reported from Pakistan, China, India, Bangladesh, Kazakhstan, Kenya, and Iran ( Figure 2).
The obtained cox1 partial fragments of ticks were submitted to GenBank under accession numbers: OQ860250 (Ha.

Discussion
Various ticks and their associated pathogens ideally propagate in Pakistan's humid and variable climatic conditions (Karim et al., 2017;Ali et al., 2019Ali et al., , 2020Ali et al., , 2021Obaid et al., 2023). Microbiota belonging to different bacterial genera have been detected in different tick species in Pakistan (Karim et al., 2017;Ali et al., 2021;Alam et al., 2022;Khan Z. et al., 2022;Khan S. M. et al., 2023;Numan et al., 2022). Some serological surveys of Q-fever in small ruminants, large ruminants, rodents, and humans have been reported from Pakistan (Ahmed, 1987;Ali et al., 2022a). There is limited information available regarding the molecular characterization of C. burnetii and thus it remains an ignored zoonotic disease in the country. The association of Coxiella spp. with different ticks infesting various hosts has been reviewed globally (Guatteo et al., 2011); therefore, in this study, we summarized this association of Coxiella spp. with different ticks in Asia. Nine tick species including Ha. cornupunctata, Ha. bispinosa, Ha. montgomeryi, Ha. danieli, Hy. anatolicum, Hy. dromedarii, R. haemaphysaloides, R. microplus, and A. persicus infesting goats, sheep, camels, cattle, wild mice, and domestic fowls were genetically characterized. In addition, this is the first report regarding the molecular detection and phylogenetic positioning of Coxiella spp. associated with ticks in Pakistan. Overall, C. burnetii, two Coxiella endosymbionts, and five undetermined Coxiella sp. were genetically characterized based on GroEL fragments in various tick species.
Environmental factors such as humidity and temperature mainly affect the distribution of ticks, TBDs, and their zoonotic threats to human and animal health (Léger et al., 2013). Since the current study area's existing environmental and climatic conditions are favorable for tick infestation and propagation of various pathogens (Aiman et al., 2022;Ali et al., 2023), many ticks were collected during this survey. Contrary to previous studies, Ha. cornupunctata tick was more prevalent than other tick species such as R. microplus and Hy. anatolicum in Pakistan (Karim et al., 2017;Ali et al., 2019Ali et al., , 2021Khan Z. et al., 2022). It may be due to examining different hosts, such as goats, sheep, and wild mice attributing a closed association with this species.

FIGURE
Phylogenetic tree based on cox fragments of tick species. The sequence of Alveonasus lahorensis (KX ) was used as an outgroup. The levels of bootstrap support (≥ %) for phylogenetic groupings are given at each node. The obtained sequences are represented with bold and underlined fonts.

FIGURE
Phylogenetic tree based on GroEL fragments of Coxiella spp. detected in tick species. The sequence of Legionella jordanis (LR ) was used as an outgroup. The levels of bootstrap support (≥ %) for phylogenetic groupings are given at each node. The obtained sequences are represented with bold and underlined fonts.
The age of the host is a significant factor to tick infestation. According to previous reports, a high tick burden was recorded on adult hosts compared with young ones Kamran et al., 2021;Khan Z. et al., 2022). Large body surfaces and free grazing practices of adult animals make them more vulnerable due to high tick infestation. In contrast, the robust immune system, less grazing, and low body surface of the younger hosts contribute to less tick infestation (Swai et al., 2005). Female hosts were highly tick infested compared with the male hosts, which is consistent with previous findings . Higher levels of progesterone and prolactin hormones in females make them susceptible to tick infestation (Anderson et al., 2013;Ahmed et al., 2023). The higher levels of progesterone and prolactin hormones may increase the susceptibility of females to tick's infections (Lloyd, 1983;Ahmed et al., 2023). Additionally, in the current study, ticks were predominantly reported in summer (June-August) compared with other seasons because the warm and humid climatic conditions in the region provide a suitable environment for the development of all stages of ticks (Ali et al., 2019. The comparatively wide host range noted for different Haemaphysalis, Hyalomma, and Rhipicephalus ticks may be due to frequent practices such as putting various hosts in the same shelter and over-crowded livestock and concurrent grazing in the survey area. Major consequences have been revealed in the epidemiology of Q-fever upon the molecular detection of C. burnetii DNA in ticks collected from the environment, domestic and wild animals (Yessinou et al., 2022). It has been observed that ticks may transmit the Q-fever agent and pollute the environment as well as the host's body in Pakistan (Ullah et al., 2019). The association between different ticks and C. burnetii and its transstadially and transovarially transmission has been reported, suggesting the Qfever transmission from infected to healthy animals through blood .
/fmicb. . meal (Gong et al., 2020). In this study, molecular detection of Coxiella spp. varied in the aforementioned seven tick species collected from various hosts. A high prevalence of Q-fever in camels in this study may be attributable to the camels' vulnerability regarding C. burnetii infection or camel tick competence as a reservoir for this pathogen (Gumi et al., 2013). Common reservoirs for C. burnetii are small ruminants that may excrete a diverse number of these bacteria in their birth byproducts (placenta). Coxiella spp. were highly detected in ticks collected from small ruminants (goats and sheep), and these findings agreed with the previous serosurvey conducted in Pakistan (Ullah et al., 2019). Coxiella sp. detected in A. persicus ticks collected from domestic fowls suggest that different soft ticks may also be investigated as host reservoirs for various undetermined Coxiella spp., as reported in other studies (Trinachartvanit et al., 2018).
In the current study, phylogenetic analysis via cox1 fragments of nine different tick species revealed a close evolutionary relationship with the same species reported from Pakistan, China, India, Bangladesh, and Iran, and these findings were supported by previous studies (Ahmad et al., 2022;Alam et al., 2022;Ali et al., 2022a;Khan S. M. et al., 2023). Phylogenetic analysis of Coxiella spp., detected in different tick species, showed close association with their respective species reported from the same or different tick species and humans. This association of Coxiella spp. may be due to the close interaction of infested animals with humans, which enhances zoonotic infections such as Q-fever in humans. Sofar neglected surveillance of Coxiella spp. in the region demands immediate attention to its pathogenic consequences.

Conclusion
Coxiella spp. were molecularly detected in ticks infesting goats, sheep, camels, cattle, wild mice, and domestic fowls and were confirmed through sequencing for the first time in Pakistan. Further research is essential to investigate any potential health risks due to these agents. The veterinarian livestock holders and farm workers lack knowledge regarding the epidemiology of Q-fever and its causative agents in Pakistan. Livestock holders should be adequately educated regarding Q-fever prevention and management practices because the occurrence of this agent can lead to long-term environmental contamination, which is a potential threat to animals and humans. Consequently, effective measures associated with Q-fever must be implemented, including limiting contact between herds, quarantining newly purchased animals, and using disinfectants that can reduce the spread of infection and possible transmission to humans.

Institutional review board statement
The Advance Studies and Research Board (ASRB: Dir/A&R/AWKUM/2022/9396) of the Department of Zoology, Abdul Wali Khan University Mardan, Pakistan, approved prior consent for this study. Additionally, permission was taken from the owners of the animals to observe hosts and ticks collection. All the rules regarding animal welfare regulations were followed while handling the animals.

Ethics statement
The animal study was reviewed and approved by the Advance Studies and Research Board (ASRB: Dir/A&R/AWKUM/2022/9396) of the Department of Zoology, Abdul Wali Khan University Mardan, Pakistan, approved prior consent for this study. Written informed consent was obtained from the owners for the participation of their animals in this study.

Author contributions
AAli designed the study. AAli, MMA, TT, SBK, and AAlo carried out the experiments of the study. AAli, MN, ZUI, GR, MKO, SBK, and SU collected the tick samples and performed the experiments. AAli, MKO, and MN performed the phylogenetic and statistical analyses. All authors have read and agreed to the published version of the manuscript.